1. Field of the Invention
The subject of the present invention is nucleic acid sequences derived from the genome of Salmonella Typhi, and their use, in particular, for the in vitro diagnosis of the presence of bacteria of the Salmonella genus in a biological sample likely to contain them, and more particularly in foodstuffs.
2. Discussion of the Background
At present, foodstuffs are of growing importance. For social reasons, transformed foodstuffs, ready for consumption, continue to create an increasing demand on the part of the buyers. The result of this is that the sources of production and display of these products have been modified considerably during the last fifteen years (mass manufacture in specialised factories; marketing in packaged units, usually in a plastic wrapping).
For reasons of public health, the manufacturing technologies and the products themselves are subjected to stricter and stricter measures of health supervision. In the framework of bacteriological controls, the bacteria responsible for toxic infections of foodstuffs are investigated systematically, and among them priority is given to the Salmonella. Moreover, the health standards are very clear for this bacterial genus: absence of Salmonella in 25 grams of product.
From the point of view of taxonomy, the Salmonella genus belongs to the family of the Enterobacteriaceae. This genus includes a single and unique species, S. enterica, which may be subdivided into even sub-species. Within each of these sub-species, it is possible to identify a large number of serotypes with the aid of sera directed against the polysaccharide 0 antigens and the flagellar H antigens. In 1989, 2267 serotypes of Salmonella were known.
The Salmonella are entero-invasive bacteria, which are pathogenic for man and animals. Their pathogenic potency is linked to their capacity to invade the intestinal epithelium. This stage can be limited to the mucosal membranes in the case of a toxic infection for example, or be followed by systemic dissemination (the case of typhoid fever). The contamination of the host by Salmonella occurs by the mouth in the very great majority of cases. This explains why the Salmonella are systematically investigated in the context of bacteriological controls of biological samples.
The standard method of systematic investigation of Salmonella, recommended by the Association Francaise de Normalisation (AFNOR) comprises: the placing in culture of the product to be analysed in two different enrichment media, incubated at two different temperatures and isolated on two different selective media (this step is often preceded by a "revivification" step in a nutrient broth); subculturing of at least 6 colonies on media for rapid identification; finally, serological typing of the strain. Even though the AFNOR protocol must be scrupulously followed during an official expert investigation, it can be imagined that it is difficult to apply during systematic controls on account of the time needed to carry out the investigation (at least one week) and its cost.
Novel methods of investigation of Salmonella are based either on enzymatic assays or on the use of nucleic acid probes. It must be emphasized that in the two cases a culture step on a rich medium and a subculture in an enrichment medium are recommended, and even essential.
The immuno-enzymatic assays which use monoclonal antibodies are easy to implement, give results in four to six hours and are available at a reasonable price. Their great disadvantage lies in the fact that they often give false positive reactions and sometimes false negative reactions. The consequences of such false reactions are considerable in both cases: if a false positive reaction is produced, there will be seizure of the product and the initiation by the analytical laboratory of a process to isolate a Salmonella which does not exist; if a false negative reaction is produced, the product will be marketed with the risks which that entails for public health.
A recent study involving 250 strains of Salmonella and 75 bacterial strains not belonging to the Salmonella genus produced 17 false positive reactions and 2 false negative reactions (D'AOUST, J. Y. (1987): "Efficacy of the immunoenzymatic kit BIOENZABEAD for the screening of Salmonella spp. in foodstuffs", Microorganisms and Foodstuffs: Rapid Procedure, industrial control. Colloquium of the French Society of Microbiology, Paris). Since this study was performed on pure cultures, it can be imagined that the number of false reactions would have been much larger with poly-microbial products (microbial competition; risk of antigenic cross-reactions).
The nucleic acid probes are constituted by a genomic DNA fragment (FITTS, R. et al. (1983), "DNA-DNA Hybridization Assay For Detection of Salmonella spp". In Foods, Applied and Environmental Microbiology, 46, 1146-1151). They are reputed to be specific. Several kits exist on the market. In fact, it appears that these probes present two major disadvantages: their lack of specificity according to the results communicated by users (cross-reaction with the Citrobacter, for example) and their lack of sensitivity (threshold limit of detection: 10.sup.5 Salmonella; or according to some authors, 2.5 .mu.g of DNA, i.e. about 10.sup.7 bacteria).